1. Project Summary / Abstract Invasive fungal infections (IFI) are a leading cause of death in the growing number of immunocompromised patients, and successful therapy is notoriously difficult. Leading FDA-approved antifungal classes are limited by inadequate clinical efficacy, which is often due to dose-limiting toxicities, emerging resistance, drug-drug interactions, and the need for therapeutic monitoring. New antifungals with robust activity against a broad spectrum of pathogens, minimal susceptibility to resistance, and limited side effects are needed. Amphotericin B (AmB) demonstrates dose-dependent killing, is fungicidal, has exceptionally broad spectrum, and has no reported development of resistance. However, the commercially available forms of AmB, including AmBisome, have both acute and chronic toxicities that preclude safe use at high doses. This toxicity hinders realization of the full clinical potential of AmB. Our goal is to develop a chemically modified AmB derivative with a broad spectrum of robust fungicidal activity, lack of resistance, and, most importantly, limited toxicity. This will enable clinicians to safely employ high-dose treatment protocols to more effectively treat IFI. Overturning half a century of prior thinking, we found that AmB primarily kills both fungal and human cells by simply binding ergosterol and cholesterol, respectively. Guided by this insight, we recently designed a new AmB derivative, C2?epiAmB, which selectively binds ergosterol over cholesterol. Accordingly, C2?epiAmB retains good fungicidal activity against many pathogens, and is non-toxic to human primary renal epithelial cells (hRECs), mice, and rats at the highest doses tested. However, C2?epiAmB also has important limitations with respect to potency and pathogen scope. Earlier studies from our labs identified AmB derivatives bearing urea motifs at C16 which show increased antifungal potency but retain unacceptable toxicities. In this research program, we will combine the toxicity-eliminating C2? modification in C2?epiAmB with efficacy-promoting urea modifications at C16 to develop a new class of hybrid polyene fungicidal agents that are both non-toxic and highly effective in eradicating IFI. A representative hybrid derivative that we recently synthesized, C2'epiAmBAU, has excellent potency against a series of important pathogens and minimal toxicity in hRECs. Building on these and many other encouraging preliminary results, we now plan to synthesize a family of C2?epiAmBUreas and extensively characterize them in state-of-the-art biophysical, mechanistic, resistance, efficacy, and toxicity studies, to identify the most promising candidates for enabling a new ?high-dose? clinical paradigm for better treating IFI. To accomplish all these goals, we have assembled a world-class multidisciplinary team of experts in chemical synthesis, antifungal development, pharmacokinetics, molecular mycology, and the clinical management of IFI. At the end of this proposal, we will be positioned for IND-enabling studies with a potentially transformative new antifungal agent.